Bacteriocins are a large class of genetically encoded antimicrobial peptides in which molecular diversity may be introduced by post-translational modifications. These compounds display diverse and often novel mechanisms of cytotoxicity (2, 3). Ribosomally synthesized peptide antibiotics are amenable to structural variation via site-directed mutagenesis providing access to analogs for structure-function studies. The biosynthesis of the lantibiotics produced by Gram-positive bacteria has long intrigued microbiologists, biochemists, and chemists (1). As illustrated in FIG. 1 for the lantibiotic, lacticin 481 (4), these compounds contain the unusual cyclic thioether amino acids lanthionine (Ln) and/or methyllanthionine (MeLn) as well as 2,3-didehydroalanine (Dha) and (Z)-2,3-didehydrobutyrine (Dhb). The widespread use of the prototypic lantibiotic nisin as an alternative to chemical reagents in food preservation in more than 80 countries for over 40 years without development of significant resistance (5) has spurred research activities directed at understanding lantibiotic biogenesis.
Genetic investigations have indicated that lantibiotics are ribosomally synthesized as precursor peptides (prepeptides), which subsequently undergo post-translational modifications (1). These modifications can include one or more dehydrations and one or more cyclizations, often forming lanthionine or methyllanthionine rings.
In vivo genetic engineering studies aimed at producing novel lantibiotics have focused on site directed mutagenesis of the genes for the precursor peptides (6-9). These investigations have uncovered several limitations including loss of lantibiotic production (10) (11-14). In vitro evaluation of the substrate specificity of purified lantibiotic synthetases can overcome these limitations and allow a detailed study of the molecular logic underlying the formation of the fused cyclic structures. However, despite much effort since the first sequencing of lantibiotic gene clusters (15), the complex biosynthetic process has not proven amenable to in vitro reconstitution.
Many lantibiotics can be grouped as Class I (LanB LanC type as defined herein) or Class II (LanM type as defined herein). Sen et al. disclose that for LanB LanC type lantibiotics such as nisin, no experimental evidence consistent with a dehydration function for LanB proteins has been reported to date (Sen A K et al., 1999, Eur. J. Biochem 261:524-532). Sen et al. further disclose that direct proof for the role of LanB awaits the in vitro dehydration of precursor peptides using the purified enzyme and that problems have been encountered by them and others (Kupke T and Gotz F, 1996, Antonie Van Leeuwenhoek 69:139-150) in various attempts.
In the context of another LanB LanC type lantibiotic, epidermin, Kupke and Gotz disclose that in incubation experiments, EpiC did not react with EpiA (the precursor peptide for epidermin), proepidermin, or with oxidative decarboxylated peptides) despite using various assay conditions (Kupke and Gotz, 1996, J. Bacteriology 178(5):1335-1340). In particular, they disclose that lanthionine formation was not investigated since no dehydrated precursor peptides were available from attempts to examine whether EpiC catalyzes the dehydration of serine and threonine residues of EpiA. In further disclosing the future intention to analyze “whether the catalytic function (dehydration of hydroxy amino acid residues or thioether formation) of EpiB and EpiC depends on (new) cofactors,” the implication is that such a useful catalytic function is not yet achieved.
Also in the context of epidermin, Peschel et al. disclose that purified and crude versions of EpiB were used in an in vitro assay for modifications of the purified epidermin precursor (Peschel A et al., 1996, FEMS Micribiology Letters 137:279-284). Despite the fact that the assay conditions were extensively varied in the presence of several potential cofactors and trace elements, no modification of the precursor peptide was detected.
In reviewing the lantibiotic field in the context of LanB LanC proteins, Jack and Jung disclose that “to date, it has not been possible to obtain lantibiotics in vitro using these proteins, and this is clearly one of the great challenges in lantibiotic research” (Jack R W, Jung G, Current Opinion in Chemical Biology 2000, 4:310-317). Sahl and Bierbaum disclose that the N-terminus of LanM enzymes does not display any similarity to the LanB proteins and that the role of LanM proteins in fulfilling the function of LanC or in being involved in the dehydration reaction is a hypothesis that remains to be proven experimentally (Sahl H-G, Bierbaum G, 1998, LANTIBIOTICS: Biosynthesis and Biological Activities of Uniquely Modified Peptides from Gram-Positive Bacteria, Annual Review of Microbiology, Vol. 52, pp. 41-79). Similar to the situation for LanB LanC type lantibiotics, apart from the present invention we are unaware of any prior indication of a successful in vitro biosynthesis for LanM type lantibiotics and therefore, for any lantibiotics.
The ability to successfully develop methods and generate useful compounds, for example any lantibiotics and variants thereof by an in vitro approach, provides an alternative to in vivo approaches. Furthermore, an in vitro approach can lead to different or improved compounds such as lantibiotics. The ability to achieve in vitro dehydration and cyclization of substrates would generally represent a significant advance.